Amphoteric liposomes and their use

ABSTRACT

Amphoteric liposomes are proposed, which comprise positive and negative membrane-based or membrane-forming charge carriers as well as the use of these liposomes.

BACKGROUND OF THE INVENTION

The invention relates to amphoteric liposomes, which simultaneouslycomprise positive and negative membrane-based or membrane-forming chargecarriers as well as to the use of these liposomes.

The concept of lipids comprises three classes of natural products, whichcan be isolated from biological membranes: phospholipids, sphingolipidsand cholesterol with its derivatives. However, it also comprisessynthetically produced materials with similar characteristics. Asrepresentatives of these, diacyl glycerols, dialkyl glycerols,3-amino-1,2-dihydroxypropane esters or ethers and also N,N-dialkylaminesare mentioned.

These substances are of technical interest for the preparation ofliposomes. One of the uses of these liposomes is as a container foractive ingredients in pharmaceutical preparations. For this purpose, anefficient and stable packaging of the cargo, compatibility with bodyfluids and a controllable and optionally site-specific release of thecontent are desirable.

It is a disadvantage that it is difficult to combine the tworequirements. The tighter and more stable the packaging, the moredifficult it is to release the enclosed active ingredient once again.For this reason, liposomes were developed, which change their propertiesin reaction to external stimuli. Heat-sensitive and pH-sensitiveliposomes are known. The pH-sensitive liposomes are of special interest,since this parameter may change under physiological circumstances, suchas during the endocytotic absorption of a liposome in cells or duringpassage through the gastrointestinal tract. According to the state ofthe art, pH-sensitive liposomes comprise, in particular, cholesterolhemisuccinate (CHEMS).

Cholesterol hemisuccinate is used in admixture with phosphatidylethanolamine for the preparation of pH-sensitive liposomes (Tachibana etal. (1998); BBRC 251:538-544, U.S. Pat. No. 4,891,208). Such liposomescan be endocytized by many cells and in this way are able to transportcargo molecules into the interior of cells, without injuring theintegrity of the cellular membrane.

The anionic character of CHEMS is a significant disadvantage. Theliposomes, prepared with it, have an overall negative charge andabsorbed by cells only with a low efficiency. Therefore, in spite of thetransfer mechanism described above, they are hardly suitable fortransporting macromolecules into cells.

Cationic liposomes with the highest possible and constant surface chargeare used to transport active ingredients into cells (transfection). Theoverall positive charge of such particles leads to an electrostaticadhesion to cells and, consequently, to an efficient transport intocells. The use of these compounds and of the liposomes, producedtherewith is, however, limited to in vitro or ex vitro uses, since suchpositively charged liposomes form uncontrolled aggregates with serumcomponents.

The limitation to very few pK values, generally to that of the carboxylgroup in the cholesterol hemisuccinate (approximately 4.5) is adisadvantage of the pH-sensitive liposomes, which are availableaccording to the state of the art. A further disadvantage of thecompounds is the limitation to negative charge carriers. These are notsuitable for binding nucleic acids and, frequently also, proteinsefficiently.

Cationic liposomes show good bonding of nucleic acids and proteins andare in a position to bring these active ingredients into cells. It is adisadvantage that they cannot be used for in vivo applications.

SUMMARY OF THE INVENTION

It was therefore an objective to produce the liposomal structures, which

-   i) permit an efficient inclusion of active in agents,-   ii) can transport these active ingredients into biological cells,-   iii) are compatible with use under in vivo conditions and-   iv) can be produced simply and inexpensively.

The inventive object is accomplished by amphoteric liposomes, whichcomprise at least one positive and at least one negative charge carrier,which differs from the positive one, the isoelectric point of theliposomes being between 4 and 8. This objective is accomplished owing tothe fact that liposomes are prepared with a pH-dependent, changingcharge.

Liposomal structures with the desired properties are formed, forexample, when the amount of membrane-forming or membrane-based cationiccharge carriers exceeds that of the anionic charge carriers at a low pHand the ratio is reversed at a higher pH. This is always the case whenthe ionizable components have a pKa value between 4 and 9. As the pH ofthe medium drops, all cationic charge carriers are charged more and allanionic charge carriers lose their charge.

The following abbreviations are used in connection with the invention:

CHEMS cholesterol hemisuccinate PC phosphatidyl choline PE phosphatidylethanolamine PS phosphatidyl serine PG phosphatidyl glycerol Hist-Cholhistidinyl cholesterol hemisuccinate

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 a microscopic image of transfection into HeLa cells withamphoteren liposomes;

FIG. 1 b microscopic image of transfection into CHO cells withamphoteren liposomes.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The membrane-forming or membrane-based charge carriers have thefollowing general structure of an amphiphile:

-   charge group—membrane anchor-   The naturally known systems or their technically modified forms come    into consideration as membrane anchors. These include, in    particular, the diacyl glycerols, diacyl phosphoglycerols    (phospholipids) and sterols, but also the dialkyl glycerols, the    dialkyl- or diacyl -1-amino-2,3-dihydroxypropanes, long-chain alkyls    or acyls with 8 to 25 carbon atoms, sphingolipids, ceramides, etc.    These membrane anchors are known in the art. The charge groups,    which combine with the these anchors, can be divided into the    following 6 groups:-   Strongly cationic, pKa>9, net positive charge: on the basis of their    chemical nature, these are, for example, ammonium, amidinium,    guanidium or pyridinium groups or timely, secondary or tertiary    amino functions.-   Weakly cationic, pKa<9, net positive charge: on the basis of their    chemical nature, these are, in particular, nitrogen bases such as    piperazines, imidazoles and morpholines, purines or pyrimidines.    Such molecular fragments, which occur in biological systems,    preferably are, for example, 4-imidazoles (histamine), 2-, 6-, or    9-purines (adenines, guanines, adenosines or guanosines), 1-, 2-or    4-pyrimidines (uraciles, thymines, cytosines, uridines, thymidines,    cytidines) or also pyridine-3-carboxylic acids (nicotinic esters or    amides).-   Nitrogen bases with preferred pKa values are also formed by    substituting nitrogen atoms one or more times with low molecular    weight alkane hydroxyls, such as hydroxymethyl or hydroxyethyl    groups. For example, aminodihydroxypropanes, triethanolamines,    tris-(hydroxymethyl)methylamines, bis-(hydroxymethyl)methylamines,    tris-(hydroxyethyl)methylanines, bis-(hydroxyethyl)methylarnines or    the corresponding substituted ethylamines.-   Neutral or zwitterionic, at a pH from 4 to 9: on the basis of their    chemical nature, these are neutral groups, such as hydroxyls,    amides, thiols or zwitterinonic groups of a strongly cationic and a    strongly anionic group, such as phosphocholine or aminocarboxylic    acids, aminosulfonic acids, betaines or other structures.-   Weakly anionic, pKa>4, net negative charge: on the basis of their    chemical nature, these are, in particular, the carboxylic acids.    These include the aliphatic, linear or branched mono-, di- or    tricarboxylic acids with up to 12 carbon atoms and 0, 1 or 2    ethylenically unsaturated bonds. Carboxylic acids of suitable    behavior are also found as substitutes of aromatic systems.-   Other anionic groups are hydroxyls or thiols, which can dissociate    and occur in ascorbic acid, N-substituted alloxane, N-substituted    barbituric acid, veronal, phenol or as a thiol group.-   Strongly cationic, pKa<4, net negative charge: on the basis of their    chemical nature, these are functional groups such as sulfonate or    phosphate esters.-   Amphoteric charge carriers, pI between 4.5 and 8.5, net positive    charge below the pI, net negative charge above the pI: on the basis    of their chemical nature, these charge carriers are composed of two    or more fragments of the groups named above. For carrying out the    invention, it is, initially, immaterial whether the charged groups    are on one and the same membrane anchor or if these groups are on    different anchors. Amphoteric charge carriers with a pI between 5    and 7 are particularly preferred for implementing the invention.

Strongly cationic compounds are, for example:

-   DC-Chol 3-β-[N-(N′,N′-dimethylmethane) carbamoyl] cholesterol,-   TC-Chol 3-β-[N-(N′, N′, N′-trimethylaminoethane) carbamoyl    cholesterol-   BGSC bisguanidinium-spermidine-cholesterol-   BGTC bis-guadinium-tren-cholesterol,-   DOTAP (1,2-dioleoyloxypropyl)-N,N,N-trimethylammonium chloride-   DOSPER (1,3-dioleoyloxy-2-(6-carboxy-spermyl)-propylarnide-   DOTMA (1,2-dioleoyloxypropyl)-N,N,N-trimethylamronium chloride)    (Lipofectin®)-   DORIE (1,2-dioleoyloxypropyl)-3-dimethylhydroxyethylammonium bromide-   DOSC (1,2-dioleoyl-3-succinyl-sn-glyceryl choline ester)-   DOGSDSO (1,2-dioleoyl-sn-glycero-3-succinyl-2-hydroxyethyl disulfide    omithine)-   DDAB dimethyldioctadecylammonium bromide-   DOGS ((C18)₂GlySper3⁺) N,N-dioctadecylamido-glycol-spermin    (Transfectam®) (C18)₂Gly⁺ N,N-dioctadecylamido-glycine-   CTAB cetyltrimethylarnmonium bromide-   CpyC cetylpyridinium chloride-   DOEPC 1,2-dioleoly-sn-glycero-3-ethylphosphocholine or other    O-alkyl-phosphatidylcholine or ethanolamines,-   amides from lysine, arginine or omithine and phosphatidyl    ethanolarnine.

Examples of weakly cationic compounds are: His-Cholhistarninyl-cholesterol hemisuccinate, Mo-Cholmorpholine-N-ethylamino-cholesterol hemisuccinate or histidinyl-PE.

Examples of neutral compounds are: cholesterol, ceramides, phosphatidylcholines, phosphatidyl ethanolamines, tetraether lipids or diacylglycerols.

Examples of weakly anionic compounds are: CHEMS cholesterolhemisuccinate, alkyl carboxylic acids with 8 to 25 carbon atoms ordiacyl glycerol hemisuccinate. Additional weakly anionic compounds arethe amides of aspartic acid, or glutarnic acid and PE as well as PS andits amides with glycine, alanine, glutamine, asparagine, serine,cysteine, threonine, tyrosine, glutamic acid, aspartic acid or otheramino acids or aminodicarboxylic acids. According to the same principle,the esters of hydroxycarboxylic acids or hydroxydicarboxylic acids andPS are also weakly anionic compounds.

Strongly anionic compounds are, for example: SDS sodium dodecyl sulfate,cholesterol sulfate, cholesterol phosphate, cholesteryl phosphocholine,phosphatidyl glycerols, phosphatid acids, phosphatidyl inositols, diacylglycerol phosphates, diacyl glycerol sulfates, cetyl phosphate orlyosophospholipids.

Amphoteric compounds are, for example,

-   Hist-Chol Nα-histidinyl-cholesterol hemisuccinate,-   EDTA-Chol cholesterol ester of ethylenediamine tetraacetic acid-   Hist-PS Nα-histidinyl-phosphatidylserine or N-alkylcarnosine.

The inventive liposomes contain variable amounts of suchmembrane-forming or membrane-based amphiphilic materials, so that theyhave an amphoteric character. This means that the liposomes can changethe sign of the charge completely. The amount of charge carrier of aliposome, present at a given pH of the medium, can be calculated usingthe following formula:z=Σni((qi−1)+10^((pK−pH))/(1+10^((pK−pH)))in which

-   qi is the absolute charge of the individual ionic groups below their    pK (for example, carboxyl=0, simple nitrogen base=1, phosphate group    of the second dissociation step=−1, etc.)-   ni is the number of these groups in the liposome.

At the isoelectric point, the net charge of the liposome is 0.Structures with a largely selectable isoelectric point can be producedby mixing anionic and cationic portions.

The structures can also be constructed so that, in particular, as the pHdrops, the charge on the molecule as a whole is actually changed fromnegative to positive. Such a reversal of charge is advantageousparticularly when the liposomes, produced with these structures, are tobe used in physiological interrelationships. Only liposomes with anoverall negative charge are compatible with blood and serum components.A positive charge leads to aggregations. Liposomes with a positivecharge are, however, very good fusogenically and can transport activeingredients into cells. A pH-dependent reversal of charge thereforepermits compounds to be constructed, which are compatible with serumbecause they have a negative charge; however, after their endocytoticabsorption, their charge is reversed and they become fasogenic only inthe cell.

In a preferred embodiment of an embodiment of the invention, theamphoteric liposomes have an isoelectric point between 5 and 7.

The invention also relates to amphoteric liposomes, which comprise atleast one amphoteric charge carrier, the amphoteric charge carrierhaving an isoelectric point of between 4 and 8.

In a preferred variation, the amphoteric charge carrier of the liposomeshas an isoelectric point of between 5 and 7.

The invention also relates to amphoteric liposomes, the liposomescomprising at least one amphoteric charge carrier and an anionic and/orcationic charge carrier.

It is appropriate that, in a preferred variation, the amphotericliposomes have an isoelectric point between 5 and 7.

In a special variation of the invention, the inventive liposomescomprise phosphatidyl choline, phosphatidyl ethanolamine, diacylglycerol, cholesterol, tetraether lipid, ceramide, sphigolipid, and/ordiacyl glycerol. However, the preparation of the liposomes can, ofcourse, be carried out with many lipid combinations of the inventiveteachings. For examples, liposomes can be synthesized using a largeamount of CHEMS (about 40%) and a smaller amount of DOTAP (about 30%).At the pK of the carboxyl group of the CHEMS, the negative charge ofthis component is already suppressed so far, that the positive chargecarrier predominates overall. An alternative formulation is the mixingof CHEMS with HisChol the stronger charging of the positive chargecarrier HisChol going along synergistically with the discharging of thenegative CHEMS.

If Hist-Chol, which in itself is amphoteric, is incorporated in aneutral membrane of, for example, phosphatidyl choline, an amphotericliposome with an isoelectric point, which largely corresponds to that ofHist-Chol, also results.

It is known to those skilled in the art how the important parameters canbe adapted by manifold variations of the inventive teachings:

-   (i) the charge density of the liposomes at the end points of the of    the charge reversals by the amount and the pKa values of the charge    carriers used,-   (ii) the slope of the charge reversal curve by the ratio of the two    charge carriers, by their absolute amounts and by an optimally    synergistic effect of two complementary pH-sensitive lipids and-   (iii) the passing of the zeta potential through zero due to the    ratio of the two charge carriers or also due to the position of the    pK value or values.

In a further variation of the invention, the liposomes have an averagesize of between 50 and 1000 nm, preferably of between 70 and 250 nm andparticularly between 60 and 130 nm. The amphoteric liposomes aresynthesized by methods known in the art, such as the injection ofethanol into a lipid solution in an aqueous buffer, by hydrating drylipid films or by detergent dialysis. The size of the liposomes canvary, generally between 50 nm and 10,000 nm. Homogeneous populations canbe produced by high-pressure homogenization or by extrusion.

In a preferred variation of the invention, the liposomes comprise anactive ingredient.

Advisably, in a preferred variation, the active ingredient is a protein,a peptide, a DNA, an RNA, an antisense nucleotide and/or a decoynucleotide.

In a farther preferred variation of the invention, at least 80% of theactive ingredient in the interior of the liposome.

The invention also relates to a method for charging a liposome withactive ingredient, a defined pH being used for the encapsulation and thepH being adjusted to a second value for separating the unbound material.

The invention furthermore also relates to a method for charging aliposome with active ingredient, the liposomes being permeabilized andclosed at a defined pH.

The invention also relates to the use of the liposomes for thepreparation of nanocapsules by depositing polymers or polyelectrolyteson the lipid layer. Such substances can be precipitated once or severaltimes on the surface. With a repeated deposition, which optionally canbe carried out in the absence of cross-linking agents, liposomalnanocapsules of the type described in the WO 00/28972 or in theW001/64330 are formed. It is advantageous that the electrostaticinteraction with the polyelectrolyte can be interrupted when thesubstances described here are used. It is known that the interaction ofa polyelectrolyte with charge carriers of the liposomal membrane canlead to the de-mixing of membrane components and to the formation oflipid clusters. In many cases, this de-mixing is associated with apermeabilization of the liposome. The inventive substances enable thisinteraction to be switched off after the coating process. The liposomesare enclosed only sterically in the nanocapsules if the pH is increasedat this time and there no longer is any interaction between the membraneand the polyelectrolyte. Cluster formation of the lipids and thepermeabilization of the membrane, associated therewith, can thus beavoided.

The invention also relates to the use of the inventive liposomes forpackaging and releasing active ingredients. In this variation, theliposomes bring about, in particular, the efficient packaging of activeingredients, such as nucleic acids. Nucleic acids are incubated withsaid lipids particularly at a low pH (about 3 to 6). After the formationof the liposomes, nucleic acids, adhering to the outside, can be washedoff by changing to a high pH (about 7 to 9).

An analogous procedure can be used to package proteins. Advantageously,the pH of the medium is adjusted to a value here, which lies between thepI of the liposome and that of the protein. It has proven to beparticularly advantageous, if the two pI values are more than one unitapart.

In a farther variation of the invention, the liposomes are used toprepare release systems in diagnostics.

In a further preferred variation of the invention, the liposomes areused as transfection systems, that is, for bringing active ingredientsinto cells.

In a further variation of the invention, the liposomes are used for thecontrolled release of their contents by fusion or permeabilization ofthe membrane. For example, liposomes of a lipid, which by itself is notmembrane-forming, can be stabilized by the incorporation of chargecarriers, such as PE. If the charge carrier is transformed into aneutral, uncharged or zwitterionic state, the permeability of themembrane is increased. Known liposomes of the state of the art(PE/CHEMS, Tachibana et al.) permit such a permeabilization at the lowpH values, which are attained under physiological conditions only in theinterior of endosomes or during passage through the stomach. Amphotericliposomes can be produced by the measures listed above in such a manner,that their neutral point lies at any desirable pH between 4 and 9. Underthese conditions, the liposomes are permeable and can deliver cargo tothe medium.

However, the liposomal formulations can be produced, processed andstored under conditions of lesser permeability. In a preferredembodiment of the invention, liposomes are produced so that they releaseof their cargo under conditions of a physiological pH, but enclose theircargo securely at a low pH. Such liposomes are suitable particularly forthe preparation of formulations with slow release kinetics, the releasebeing initiated only by contact with body fluids and not during storageor transport.

A preferred embodiment of the inventive teaching therefore consists ofthe use of such liposomes for therapeutic purposes, especially for suchuses, which employ the specific targeting of the liposomes. The slightnonspecific binding is a prerequisite here for transporting theliposomes to the target place. In contrast to this, a high nonspecificbinding would prevent any transport of the liposomes to the targetplace. A specific binding can be attained by further measures of thestate of the art, that is, by selecting the size of the liposomes oralso by binding the ligands to the liposomal surface, which binds to atarget receptor of the cell surface. Ligands may, for example, beantibodies or their fragments, sugars, hormones, vitamins, peptides,such as arg-gly-asp (RGD), growth factors, bilirubin or othercomponents.

The preferred variation of the inventive teachings relates to the use ofthe liposomes for therapeutic or diagnostic applications under in vivoconditions. Preferably, such liposomes are ones, which have a slightnonspecific binding and, with that, a slight tendency to fuse underphysiological conditions, but are combined strongly and with a highfusion competence under changed conditions. Such liposomes areamphoteric liposomes, which have an overall anionic particle chargeunder physiological conditions and an increasingly cationic charge at apH below 6.5. Such pH values occur during the endocytosis of theliposomes into cells. Such pH values also occur in the interior oftumors and in the external layers of the skin. Low pH values can also beobtained by perfusing an organ ex vivo for a certain period of time. Ahigh binding strength and fusion competence is therefore limited tothose liposomes, which were already taken up by cells or special tissue.The binding strength and increasing fusion competence support the fusionof the liposomal membrane with the cell membrane. This event leads to adirect release of the cargo into the interior of the cell withoutreleasing components of the lysis of the endosome and, with that,endangering the cargo or cell components.

Furthermore, the use of the liposomes as a sustained release formulationand/or as a circulating depot is appropriate. The liposomes can also beused advantageously for intravenous or peritoneal application. In aparticularly preferred variation of the invention, the liposomes areused as a vector for the in vivo, in vitro and ex vivo transfection ofcells.

The inventive liposomes have several advantages. Cationically chargeableliposomes of 40 percent HisChol and PC bind the nucleic acids, such asDNA, to their membrane even under conditions of a neutral pH.Surprisingly, this binding is suppressed completely if theabove-mentioned liposomes are produced using 5 percent of PG in additionand then have amphoteric properties. However, the binding of nucleicacids to the membrane can be restored once again by decreasing the pH.The inventive liposomes are therefore particularly well suited for thepH-dependent binding of nucleic acids.

Furthermore, it was surprisingly found that a series of proteins alsobehaves in the manner described for nucleic acids. For example,antibodies bind not at a neutral pH, but under slightly acidicconditions effectively to the membrane of the inventive liposomes. Sucha behavior cannot be observed in the case of pH-sensitive liposomes froma neutral lipid and CHEMS nor from such a liposomes from a neutral lipidand HisChol. It is therefore a special property of the amphotericliposomes. Surprisingly, it was also found that inventive liposomes,contrary to the known, constitutive, cationic liposomes, are compatiblewith serum. An appropriate embodiment of the inventive teachingstherefore consists of the use of such liposomes for therapeuticproperties. It is an advantage of the liposomes that, in comparison toknown, constitutive, cationic liposomes, the nonspecific binding tocells is significantly less.

It is, however, also surprising that the fusion competence of theinventive liposomes depends on the pH of the medium. In comparison tobiological membranes of cells, the fusion competence is determined bythe lipid selected and also by the charging of the liposomes. Usually, abinding step precedes the actual fusion. However, strong binding of theliposomes to cell membranes is not always desirable and should takeplace, as described above, only under controlled conditions inparticular cells or tissue.

The liposomes can therefore by used to construct liposomal vectors forthe transport of active ingredients into cells. All materials, which donot form micelles, come into consideration as active ingredients.Water-soluble materials are particularly suitable as active ingredients.They include many proteins and peptides, especially antibodies orenzymes or antigens, all nucleic acids, independently of their molecularweight and their derivation from RNA or DNA. However, they include alsoother biological macromolecules, such as complex sugars, naturalproducts and other compounds, as well as low molecular weight activeingredients of synthetic or natural origin, which otherwise cannotpenetrate through the cell membrane as barrier. With the help ofvectors, such materials can then be transported into the interior ofcells and initiate actions, which are not possible without thistransport.

Accordingly, with the help of the inventive teachings, liposomes can beprepared, the fusion and binding properties of which differ at differentpH values. Serum-compatible liposomes, which are laden with a largeamount of active ingredients and transport these into the interior ofcells, can therefore be produced in this way. Someone, skilled in theart, is able to combine elements of the inventive teachings with oneanother and, with that, produce liposomes, which are optimally suitablefor a particular purpose.

The invention is described in greater detail in the following by meansof examples without being limited to these examples.

EXAMPLE 1

Preparation and Charge Properties of Amphoteric Liposomes with ChargeCarriers, which can be Charged Positively and are Constantly ChargedNegatively

His-Chol (5 mg) and 7.8 mg of POPC and 2 mg of DPPG are dissolved in 4ml of a 1:1 (v/v) mixture of chloroform and methanol and driedcompletely in a rotary evaporator. The lipid film is hydrated with 4.3mL of the appropriate buffer (10 mM KAc, 10 mM HEPES, 150 mM NaCl, pH7.5, in a lipid concentration of 5 mM by a five-minute treatment withultrasound. Subsequently, the suspension is frozen and, after thawing,extruded several times (Avestin LiposoFast, polycarbonate filter with a200 nm pore width). For measuring the zeta potential, the finalconcentration of the liposomes is adjusted to a value of 0.2 mM. For thedilution, the buffer system, named above, is used at a pH of 7.5 or 4.2.The zeta potentials measured lie between −18 mV (at pH 7.5) and +35 mV(at pH 4.2).

EXAMPLE 2

Preparation and Charge Properties of Amphoteric Liposomes with ConstantPositive and Variable Negative Charge Carriers

POPC, DOTAP and CHEMS are dissolved in the molar ratios given below in 4mL of a 1:1 (v/v) mixture of chloroform and methanol and evaporatedcompletely in the rotary evaporator. The lipid film is hydrated with 4.3mL of the appropriate buffer (10 mM KAc, 10 mM HEPES, 150 mM NaCl, pH7.5, in a total lipid concentration of 5 mM by a five-minute treatmentwith ultrasound. Subsequently, the suspension is frozen and, afterthawing, excluded repeatedly (Avestin LiposoFast, polycarbonate filterwith a 200 nm pore width). The Table below shows the zeta potentials asa function of pH.

Composition of the liposomes in mole percent

liposome 1 POPC 50 DOTAP 40 Chems 10 liposome 2 POPC 50 DOTAP 30 Chems20 liposome 3 POPC 50 DOTAP 25 Chems 25 liposome 4 POPC 50 DOTAP 20Chems 30 liposome 5 POPC 50 DOTAP 40 Chems 10

TABLE 1 Zeta Potentials in mV Liposome Liposome Liposome LiposomeLiposome pH 1 2 3 4 5 4 44.2 38.4 34.7 31.7 16.2 5 39.9 25.6 27.2 22.13.3 6 37 21.4 16.4 2.5 −7.3 7.5 29.2 1.8 −7.9 −18.9 −34.6

The height of the zeta potential and its slope can be selected withinwhy limits by means of a suitable composition.

EXAMPLE 3

Preparation and Charge Properties of Amphoteric Liposomes with CompleteSwitchability in One Compound

His-Chol (5 mg) and 9.8 mg of POPC are dissolved in 4 ml of a 1:1 (v/v)mixture of chloroform and methanol and dried completely in a rotaryevaporator. The lipid film is hydrated with 4.3 niL of the appropriatebuffer (10 mM KAc, 10 mM HEPES, 150 mM NaCl, pH 7.5, in a lipidconcentration of 5 mM by a five-minute treatment with ultrasound.Subsequently, the suspension is frozen and, after thawing, extrudedseveral times (Avestin LiposoFast, polycarbonate filter with a 200 nmpore width). The course of the zeta potential at different pH values andionic strengths is shown in the table below (Table 2).

TABLE 2 pH Without Salt 100 mM of NaCl 4 45.6 20.2 5 26.9 2.2 6 −4.1−5.2 7 −31.4 −15.3 8 −45.7 −25.4

EXAMPLE 4

Serum Aggregation

Lipid films are prepared as in Example 1. A lipid mixture, which did notcontain any DPPG, was used as comparison sample. The lipid films werehydrated in buffer (10 mM of phosphate, 150 mM of sodium chloride, pH of7.4) and extruded as above. Human serum is diluted with an equal amountof buffer (10 nmm of phosphate, 150 mM of sodium chloride, pH of 7.4),particular components and fat being removed by centrifuging (20 minutes,13,000 rpm, 4° C.); the clear serum is filtered sterile with a filterhaving a pore width of 0.2 μm.

The liposomes, prepared above are added to the serum in concentration of1 mM and incubated for 15 minutes at 37° C. After the incubation, thesuspension of the DPPG-containing liposomes is uniformly cloudy;however, flocculation cannot be observed. The diameter of the liposomesis deternined by means of dynamic light scattering and is changed byless than 10% from that of the starting sample. The suspension of theDPPG-free liposomes clearly shows flocculation.

EXAMPLE 5

Stability of the Membrane

Aside from serum aggregation, the precipitation of an active ingredient(carboxyfluorescein, CF) in the presence of human serum was alsoinvestigated. For this purpose, POPC/DOTAP/CHEMS liposomes of differentdecomposition were prepared by the method of Example 2: POPC 100% (ascontrol), POPC/DOTAP/CHEMS 60:30:10, 60:20:20 and 60:10:30 (in mole %).Any CF, which is not enclosed, was removed by gel filtration. For themeasurement, the liposomes were diluted to 0.1 mM in serum and incubatedat 37° C. A 30 μL sample was removed at certain times and diluted to 300μL with 100 M of tris buffer, having a pH of 8.2 and the fluorescencewas measured. The 100% values were obtained by dissolving the liposomeswith 10 μL of Tritron X-100 (10% in water). The enclosed CF as afunction of time is shown in the Table below.

The liposomes lose only a little CF into the serum during the 4-hourperiod of rement. POPC/DOTAP/CHEMS 60:30:10 and 60:20:20 still containabout 75%, and POPC/DOTAP/CHEMS 60:10:30 even 100% of their original CFcontent (see 3).

TABLE 3 Time in POPC/DOTAP/CHEMS POPC/DOTAP/CHEMS POPC/DOTAP/CHEMS Min.POPC 60:30:10 60:20:10 60:10:30 0 100%  100%  100%  100% 15 91% 84% 95%107% 60 94% 81% 87% 110% 120 96% 80% 76% 105% 240 96% 80% 77% 107%

EXAMPLE 6

Binding DNA

Liposomes of the following compositions (in mole %) are prepared as inExample 1 (all data is in mole %).

A: 60 POPC 40 HisChol B: 55 POPC 40 HisChol 5 CHEMS C: 60 POPC 20HisChol 20 CHEMS

The liposomes are suspended in a concentration of 0.2 mM in buffer (10mnM of potassium acetate, 10 mM of HEPES, pH 4.2 or 7.5). A DNA solution(45 μL, 1 mg of DNA (Hering sperm, SIGMA D3159) in 1 mL of water) areadded in each case to 1 mL of the different liposomes samples and mixedquickly. After an incubation period of 15 minutes, the sample is filledup with 6 mL of the appropriate buffer and the zeta potential of theliposomes is measured (Table 4).

TABLE 4 pH 4.2 pH 7.5 Lipid −DNA +DNA −DNA +DNA A +47.6 −32.0 +2.4 −44.4B +47.8 −28.1 +0.1 −38.4 C +34.0 −28.6 −10.1 −24.7

Under the conditions of an excess of cationic charges (pH 4:2), there isa strong reversal of the charge of the particles. At a neutral pH of7.5, the CHEMS in high concentration (liposome C) can overcompensate thecharge of the HisChol and the particles have a negative zeta potential.Only slight amounts of DNA bind to such particles.

EXAMPLE 7

Binding and Detaching DNA

Liposomes having the compositions POPC/DOTAP/CHEMS in the ratio of60:15:25 and POPC/DCChol/CHEMS in the ratio of 60:15:25 (in mole %),were prepared by the method of Example 2. The binding of the DNA wascarried out at a pH of 4.2 by the method of the above example and thezeta potentials were determined. Subsequently, the pH of the samples wasadjusted to a value of 7.5 and the zeta potential was measured onceagain.

Mixture Zeta (mV) a) POPC/DCChol/CHEMS 60:15:25 (pH 4.2) −43.5(aggregate) b) POPC/DOTAP/CHEMS −43.7 c) POPC/DCChol/CHEMS −18.5 d)POPC/DOTAP/CHEMS −14.5

In the presence of DNA, a negative zeta potential is measured at a lowpH; however, the original particles were charged positively. After thechange to the neutral pH, this charge, which is due to the DNA, isdecreased. The zeta potentials approach that of the untreated liposomes(−11 mV at a pH of 7.5).

EXAMPLE 8

DNA Inclusion and Detachment of Material not Encapsulated

Two liposome formulations, having compositions of POPC60/DOTAP15/CHEMS25and POPC85/DOTAP15 respectively, are prepared as dry lipid films asdescribed above. In each case, the total amount of lipid was 4 μmoles.For hydration, Herings DNA was dissolved in 10 mM of potassium acetate,10 mM of HEPES and 100 mM of sodium chloride at a pH of 4.0. The DNA (4mg) was added directly to the lipid films. The resulting liposomes werefrozen and thawed repeatedly and subsequently extruded through a 200 nmfilter.

Each 500 μL of particles was mixed with 2.5 mL of a sucrose solution(0.8M sucrose in the above buffer, at a pH of 4.0 or 7.5). Over this,1.5 mL of a 0.5 M sucrose solution and 0.5 mL of the buffer were placed.

Liposomes were then separated by flotation from unbound DNA. After theflotation, the liposomes were removed from the buffer/0.5 M sucroseinterface. The amount of bound DNA was determined by intercalation ofpropidium iodide. The Stewart assay was used to determine the amount oflipid. Only the PC used responds in the Stewart assay. The other lipidswere not calculated by means of this value. The results are shown in theTable below (Table 5).

TABLE 5 Liposome pH 4.0 pH 7.5 POPC/DOTA/ 2 μg DNA/μg DOTAP 1.2 μgDNA/μg DOTAP CHEMS 60/15/25 POPC/DOTAP 2.3 μg DNA/μg DOTAP 2.3 μg DNA/μgDOTAP 85/15

With the amphoteric liposomes, only about half of the bound DNA floatsup after the change in pH to 7.5. This material is the actually enclosedmaterial. Similar results are obtained by digesting with DNAse

DNA cannot be detached once again from constitutively cationic liposomesby changing the pH or by additionally increasing the ionic strength andalways remains on the outside.

EXAMPLE 9

Fusion Properties

Liposomes with the following compositions are prepared as in Example 1(all data in mole %):

A) POPC 60 HisChol 40 B) POPC 55 HisChol 40 CHEMS 5 X) POPC 100 Y) POPC60 DPPG 40

The facultative cationic liposomes A or B are incubated with the neutralliposomes X or the anionic liposomes Y in the buffer (10 mM HEPES, 10 mMpotassium acetate, pH 4.2 or 7.5). The possible fusion of liposomes isanalyzed by size measurement by means of dynamic light scattering (Table6).

TABLE 6 Liposome 1 X X Y Y Liposome 2 A B A B pH 4.2 161.6 nm 191.9 nm1689.3 nm 2373.2 nm pH 7.5 191.8 nm 202.4 nm  250.0 nm  206.3 nm

The starting sizes of the liposomes were 161.8 nm at pH 4.2 and 165.9 nmat pH7.5

-   A) 183.2 nm-   X) 195.2 nm-   Y) 183.2 nm

The size of the pairs with the complementary charge (YA and YB) differsclearly from the size of the mixed suspensions with the neutral liposomeX. The extent of the interaction is determined by the magnitude of thecharge of the facultative cationic liposomes. The extent of the fusionto larger units does not depend on the fusogenic lipid PE.

EXAMPLE 10

Permeability to Macromolecules

DOPE (13.75 μmoles), 2.5 μmoles of CHEMS and 10 μmoles of HisChol aredissolved in isopropanol and the solvent is drawn off under a vacuum. Asolution (2.5 ML) of proteinase K in buffer (1 mg/mL of proteinase K, 10mM of potassium acetate, 10 mM HEPES, 150 mM of sodium chloride, pH 4.2)is added to the dried lipid film. After the film is hydrated, theliposomes formed are extruded through a 400 nm membrane. Proteinase,which is not enclosed, is removed by floatation of the liposome in thesucrose gradient. The liposomes, so produced, are incubated with 7.5 MLof buffer at a pH of 4.2 and 7.2 (buffer as above, starting pH 4.2 and8.0). After the combination, the proteinase K released is removed usinga 0.1 μm membrane. The liposomes, remaining in the filter, are thentreated with 7.5 mL of a solution of Triton X-100 in buffer (as above,pH 8.0).

All filtrates are tested for the presence of proteinase K. For thispurpose, a solution of azocasein (6 mg/mL of azocasein in 1 M urea, 200mM tris sulfate, pH 8.5) is used. This solution (500 μL) is mixed with100 μL of filtrate or buffer and incubated for 30 minutes at 37° C. Thereaction is terminated by the addition of 10% trichloroacetic acid.Precipitated proteins are removed by centrifuging. The coloration ismeasured at 390 nm (Table 7).

TABLE 7 Absorption at 390 nm pH of Incubation Triton X-100 Blank 4.2 −0.0192 4.2 + 0.2345 7.2 − 0.2210 7.2 + 0.0307

If the incubation of the liposomes is carried out at a pH of about 4.2,very little if any proteinase K is released. Only the dissolution of theliposomes with Triton X-100 leads to the release of the enzyme. If theliposomes are incubated at a pH of 7.2, the bulk of the enzyme isreleased already without the addition of the Triton and is found in thefirst filtrate. Hardly any additional enzyme is then dissolved from theliposomes by the addition of Triton.

EXAMPLE 11

Protein Binding

Liposomes, having the composition POPC50/DOTAP10/CHEMS40 (all data inmole %) are prepared as in the preceding examples. A solution of 0.26mg/mL of lysozyme in buffer (10 mM MES of pH 5.0 or pH 6.0 or 10 mM ofHEPES of pH 7.0 or pH 8.0) is used to hydrate the lipid film. After thehydration, all samples were frozen and thawed repeatedly. Subsequentlythe liposomes are homogenized by ultrasound and extruded through a 200nm filter.

The liposome suspension, so prepared, is adjusted to a pH of 4.0 by theaddition of acetic acid. Subsequently the liposomes are separated byflotation from protein, which has not been incorporated. The proportionof enclosed protein is given in the Table below (Table 8).

TABLE 8 pH during Inclusion % of Material Enclosed 5.0 4 6.0 21 7.0 758.0 80

Liposomes of the composition used show a pI of 5; the lysozyme is abasic protein with a pI of 11.5. The two partners therefore haveopposite charges at a pH between 6 and 8. An efficient inclusion in theliposomes is brought about by electrostatic attraction. Protein, notencapsulated, was removed at a pH of 4. The interaction between thepartners is cancelled at this pH.

EXAMPLE 12

Transfection Into Cells

HeLa cells or CHO cells (3×10⁵) were plated into each cavity of a 6-welltiter plate and cultured for three days. Liposomes (POPC/DOTAP/CHEMS60/30/10) were prepared in the presence of fluorescence-labeled dextran(TRITC dextran 10 mg/mL in the hydration buffer). TRITC dextran, whichhad not been incorporated, was removed by gel filtration. The liposomes,so prepared, were added to the cells and incubated for 6 hours at 37° C.Subsequently, the cells were washed twice with buffer. The absorption ofdextran was followed in the microscopic image. The results are shown inFIG. 1.

EXAMPLE 13

Ligand Binding and Transfection

Liposomes, having the composition of POPC/DOTAP/Chems/N-glutaryl-DPPE(50:10:30:10 (mole %)) are prepared as in Example 2. At the same time,they are hydrated with a solution of 3 mg/mL of TRITC-Dextran (having amolecular weight of about 4,400) in HEPES 10 mM and 150 mM of sodiumchloride at a pH of 7.5. TRITC-Dextran, which is not enclosed, isremoved by gel filtration through a Sephadex G-75 column. Activation ofthe N-glutaryl DEPPs with EDC (1-ethyl-3-(3-dimethylaminopropylcarbodiimide) (3.5 mg of EDC per 400 μL of liposome suspension) andsubsequent stirring in the dark for 5 hours brings about the binding ofthe cyclic peptide RCDCRGDCFC to the liposomal surface. The RGD peptide(250 μg in 150 μL of buffer) was then added and stirring was continuedovernight. The liposomes were separated by gel filtration from thepeptide, which had not been bound.

Human endothelium cells (HUVEC) were cultured in a special medium. Theliposomes, modified with ligand, and control liposomes without RGDligand were added as a 0.5 mM suspension to the cells. After 2 hours,the liposomes are removed and the cell chambers rinsed 3 times with PBSbuffer and viewed under the fluorescence microscope. The TRITCfluorescence of cells, which had been treated with RDG liposomes, isdistinctly more red than that of the control liposomes.

EXAMPLE 14

Pharmacokinetics (Blood Level and Organ Distribution of pH-SwitchableLiposomes)

Liposomes of POPC/Chol (60:40), POPC/Hist-Chol/Chol (60:20:20) andPOPC/DOTAP/Chems (60:10:30) (500 μL) were injected into the tail vein ofmale Wistar rats.

Liposome suspensions (50 mM) were prepared by hydrating a lipid film ofthe corresponding formulation (addition of 0.03 moles of [14]C-DPPC)with 2 mL of a solution of 1 mg [3]H-inulin in HEPES 10 mM, sodiumchloride 150 nm at a pH of 7.5). After 3 freezing and thawing cycles,the suspensions were extruded repeatedly through a 400 nm membrane(LiposoFast, Avestin). [3]H-Inulin which had not been enclosed, wasremoved by gel filtration though a G-75 Sephadex-column and subsequentconcentration over CENTRIPREP (Millipore) centrifuging units.

Liposome suspension (0.5 mL) was administered to 4 experimental animalsper formulation and blood samples were taken after 5 minutes, 15minutes, 60 minutes, 3 hours, 12 hours and 24 hours. The radioactivityof the membrane fraction and of the soluble cargo was measured byscintillation and gave the following values:

Elimination half-life times from the blood

POPC/Chol greater than 120 minutes POPC/DOTAP/Chems greater than 120minutes POPC/Hist-Chol greater than 120 minutes

With their relatively long half-life in the blood, the inventiveliposomes fulfill the basic prerequisites for a vector system. They arenot acutely toxic and not absorbed immediately by theirreticuloendothelial system. Up to the end of the experiment, the ratioof the 3[H] to the 14[C] radioactivity of the blood samples wasconstant. Release of the cargo by complement lysis therefore does nottake place in any of the cases.

1. Amphoteric liposomes comprising an active ingredient, having anisoelectric point of between 4 and 7, wherein the liposomes comprise atleast one amphipatic cationic lipid, at least one amphipatic anioniclipid, and at least about 30% neutral lipid, and wherein said liposomesare stable at pH 4.2 and pH 7.5.
 2. The amphoteric liposomes of claim 1,wherein the liposomes have an isoelectric point of between 5 and
 7. 3.Amphoteric liposomes comprising an active ingredient, wherein theliposomes comprise at least one amphipatic lipid with both a positivecharge and a negative charge, and at least about 30% neutral lipid,wherein the amphoteric liposomes have an isoelectric point of between 4and 8 and are stable at pH 4.2 and pH 7.5.
 4. The amphoteric liposomesof claim 3, wherein the liposomes further comprise at least oneamphipatic lipid with a positive charge or at least one amphipatic lipidwith a negative charge.
 5. Amphoteric liposomes of claim 3, wherein theliposomes have an isoelectric point between 5 and
 7. 6. The amphotericliposomes of claim 1, 2, 3, 4, or 5, wherein said neutral lipid isselected from the group consisting of phosphatidyl choline, phosphatidylethanolamine, cholesterol, tetraether lipid, ceramide, sphingolipid, anddiacyl glycerol.
 7. The amphoteric liposomes of claim 1 or 3, whereinthe liposomes have an average size of between 50 and 1000 nm.
 8. Theamphoteric liposomes of claim 1, wherein the active ingredient is aprotein, a peptide, a DNA, and RNA, antisense nucleotide and or a decoynucleotide.
 9. The amphoteric liposomes of claim 1, wherein at least 80percent of the active ingredient is in the interior of the liposome. 10.The amphoteric liposomes of claim 7, wherein the liposomes have anaverage size of between 70 and 250 nm.
 11. The amphoteric liposomes ofclaim 7, wherein the liposomes have an average size of between 60 and130 nm.
 12. Amphoteric liposomes of claim 1 wherein the anionic lipid isa weak anion and the cationic lipid is a strong cation and the anion ispresent in excess over the cation.
 13. Amphoteric liposomes of claim 12,wherein said weakly anionic lipid is selected from the group consistingof cholesterol hemisuccinate (CHEMS), diacyl glycerol hemisuccinate,fatty acids and phosphatidyl serine.
 14. Amphoteric liposomes of claim12, wherein said strongly cationic lipid is selected from the groupconsisting of DOTAP, DC-Chol, DORIE, DDAB, TC-Chol, DOTMA, DOGS,(C18)₂Gly⁺ N,N-dioctadecylamido-glycin, CTAB, CPyC and DOEPC. 15.Amphoteric liposomes of claim 12, wherein the anionic lipid is CHEMS ordiacylglycerol hemisuccinate, the cationic lipid is DOTAP or DC-Chol andthe neutral lipid is phosphatidylcholine.
 16. Amphoteric liposomes ofclaim 12, wherein said liposome comprises about 50 mol. % POPC, about 20mol. % DOTAP and about 30 mol. % CHEMS.
 17. Amphoteric liposomes ofclaim 12, wherein said liposome comprises about 50 mol. % POPC, about 10mol. % DOTAP and about 40 mol. % CHEMS.
 18. Amphoteric liposomes ofclaim 12, wherein said liposome comprises about 60 mol. % POPC, about 10mol. % DOTAP and about 30 mol. % CHEMS.
 19. Amphoteric liposomes ofclaim 12, wherein said liposome comprises about 60 mol. % POPC, about 15mol. % DOTAP and about 25 mol. % CHEMS.
 20. Amphoteric liposomes ofclaim 12, wherein said liposome comprises about 30 mol. % POPC, about 30mol. % DOTAP and about 40 mol. % CHEMS.
 21. Amphoteric liposomes ofclaim 12, wherein said liposome comprises about 60 mol. % POPC, about 15mol. % DC-Chol and about 25 mol. % CHEMS.
 22. Amphoteric liposomes ofclaim 1, wherein the anionic lipid is a weak anion and the cationiclipid is a weak cation.
 23. Amphoteric liposomes of claim 22, whereinsaid weakly anionic lipid is selected from the group consisting ofcholesteryl hemisuccinate (CHEMS), diacyl glycerol hemisuccinate, fattyacids and phosphatidyl serine.
 24. Amphoteric liposomes of claim 22,wherein said weakly cationic lipid is selected from the group consistingof HisChol and MoChol.
 25. Amphoteric liposomes of claim 22, whereinsaid weakly anionic lipid is CHEMS or diacylglycerol hemisuccinate, saidweakly cationic lipid is HisCRol or MoChol and the neutral lipid isphosphatidylcholine.
 26. Amphoteric liposomes of claim 22, wherein saidliposome comprises about 55 mol. % POPC, about 40 mol. % HisChol andabout 5 mol. % CHEMS.
 27. Amphoteric liposomes of claim 22, wherein saidliposome comprises about 60 mol. % POPC, about 20 mol. % HisChol andabout 20 mol. % CHEMS.
 28. Amphoteric liposomes of claim 1, wherein theanionic lipid is a strong anion and the cationic lipid is a weak cationand the cation is present in excess over the anion.
 29. Amphotericliposomes of claim 28, wherein said strongly anionic lipid is selectedfrom the group consisting of cholesterol sulphate, cholesterolphosphate, phosphatidyl glycerol, phosphatidic acid, phosphatidylinositol, cetyl phosphate.
 30. Amphoteric liposomes of claim 28, whereinsaid weakly cationic lipid is selected from the group consisting ofHisChol and MoChol.
 31. Amphoteric liposomes of claim 28, wherein saidstrongly anionic lipid is selected from phosphatidylglycerol,phosphatidic acid, or cetyl phosphate and said weakly cationic lipid isselected from HisChol or MoChol and the neutral lipid isphosphatidylcholine.
 32. Amphoteric liposomes of claim 28, wherein saidliposome comprises about 47,5 mol. % POPC, about 40 mol. % HisChol andabout 12,5 mol. % DPPG.